Malaria is caused by protozoan parasites of the genus Plasmodium. P. falciparum is the most virulent species and is responsible for the majority of both morbidity and mortality, including approximately 800,000 deaths per year that occur mainly among young children. Key to the parasite's ability to survive attack by the host's immune system is its ability to repair DNA damage. DNA double strand breaks (DSBs) are lethal if not repaired, and malaria parasites appear to be missing one of the two primary pathways used by most eukaryotes to repair such breaks. In addition, the remaining pathway requires a second DNA copy homologous to the site of the break to serve as a template for repair. Considering that malaria parasites are haploid for most of their lifecycle and therefore the majority of the genome is maintained as a single copy, how they repair DSBs remains a mystery. The long-term objectives of this proposal are to understand how parasites maintain the integrity of their genomes, and to determine how DNA repair pathways contribute to diversification of the genes encoding their primary surface antigens. To address these objectives, a regulatable, site-specific endonuclease system has been adapted for use in cultured parasites. This system enables the induction of a single DSB within a targeted site of the genome followed by the rapid and efficient isolation of the products of repair. In the first ai of the project, this system will be used to characterize the basic mechanisms employed by parasites to repair DSBs, including how error prone the process is, whether a template is required, and the role of the mismatch repair pathway in DSB repair. In the second aim, the system will be applied to investigating diversification of the var gene family. var genes encode PfEMP1, the primary malaria virulence factor. This large, multi-copy gene family undergoes rapid and continuous diversification that enables the parasite to avoid the immune system through antigenic variation. The mechanisms underlying diversification are unknown, but the process appears to involve frequent shuffling of segments, a hallmark of gene conversion events that are a product of DSB repair. The site-specific endonuclease system will be used to determine how DSB repair contributes to var gene diversification. PUBLIC HEALTH RELEVANCE: Malaria parasites utilize their basic DNA repair pathways both to survive DNA damage resulting from the human immune response and to generate diversity within the genes encoding proteins expressed on the surface of infected red blood cells. Investigations into how the parasite is able to avoid immunity or identify potential weaknesses in the parasite's ability to repair DNA damage will contribute to the development of novel disease intervention strategies.